Method for producing ergothioneine by using soybean cake powder as nitrogen source in a seed medium

Abstract

The present disclosure relates to an improved method for producing ergothioneine, comprising the steps of: (a) inoculating Pleurotus ostreatus strain CGMCC No.6232 into a seed medium, and culturing it to prepare a seed liquor, wherein the seed medium uses soybean cake powder as nitrogen source; and (b) inoculating the seed liquor into a fermentation basal medium, and then culturing it to obtain a fermentation broth of Pleurotus ostreatus mycelia. Further, any one or more members selected from NH4Cl, NH4NO3, NaCl, polyethylene glycol, folic acid, vitamin B1 (VB1), indolebutyric acid, citric acid, pyruvic acid, arginine, lysine, leucine, aspartic acid, glutamic acid, betaine, histidine, cysteine, methionine, tween, span, chitosan, Fluconazole, Miconazole, Ketoconazole, ethylenediaminetetraacetic acid (EDTA), isopropyl alcohol and dimethyl sulfoxide are added into the fermentation basal medium.

The invention claimed is: 
     
       1. A method for producing ergothioneine, comprising the steps of:
 (a) inoculating  Pleurotus ostreatus  strain CGMCC No.6232 into a seed medium, and culturing it to prepare a seed liquor, wherein the seed medium uses soybean cake powder as nitrogen source; and 
 (b) inoculating the seed liquor into a fermentation basal medium, and then culturing it to obtain a fermentation broth of  Pleurotus ostreatus  mycelia, wherein ergothioneine is accumulated in mycelial cells of  Pleurotus ostreatus  mycelia; and 
 (c) extracting ergothioneine from the mycelial cells; 
 
       wherein using soybean cake powder as the nitrogen source in step (a) results in an increased amount of the production of ergothioneine in mycelial cells of said  Pleurotus ostreatus  strain CGMCC No.6232 in step (b) as compared to using soybean meal powder with the same concentration as nitrogen source of the seed medium. 
     
     
       2. The method according to  claim 1 , wherein the culturing process in step (a) is carried out at 19-31° C. for at least 3 days, the culturing process in step (b) is carried out at 19-31° C. for at least 6 days, and the inoculation amount in step (b) is 4-20% (V/V). 
     
     
       3. The method according to  claim 1 , wherein the seed medium comprises 15-50 g/L corn flour, 5-35 g/L soybean cake powder, 20-80 U/L α-amylase, 1-6 g/L KH 2 PO 4 , 0.2-5 g/L MgSO 4 .7H 2 O, and a balance of water. 
     
     
       4. The method according to  claim 3 , wherein the content of the soybean cake powder is 15-35 g/L. 
     
     
       5. The method according to  claim 1 , wherein the fermentation basal medium comprises 10-95 g/L glycerol, 10-80 g/L casein peptone, 2-4 g/L KH 2 PO 4 , 0.5-2 g/L MgSO 4 .7H 2 O, and a balance of water. 
     
     
       6. The method according to  claim 5 , wherein the fermentation basal medium comprises 65-95 g/L glycerol. 
     
     
       7. The method according to  claim 5 , wherein the fermentation basal medium comprises 40-80 g/L casein peptone. 
     
     
       8. The method according to  claim 1 , wherein at least one member selected from the group consisting of NH 4 Cl, NH 4 NO 3 , NaCl, polyethylene glycol, folic acid, vitamin B1 (VB1), indolebutyric acid, citric acid, pyruvic acid, arginine, lysine, leucine, aspartic acid, glutamic acid, betaine, histidine, cysteine, methionine, tween, span, chitosan, Fluconazole, Miconazole, Ketoconazole, ethylenediaminetetraacetic acid (EDTA), isopropyl alcohol and dimethyl sulfoxide is added into the fermentation basal medium. 
     
     
       9. The method according to  claim 8 , wherein said at least one member is selected from the group consisting of tween, Fluconazole, Miconazole, Ketoconazole, ethylenediaminetetraacetic acid (EDTA), isopropyl alcohol and dimethyl sulfoxide. 
     
     
       10. The method according to  claim 1 , wherein at least one member selected from the group consisting of NH 4 Cl 0.5 g/L-12 g/L, NH 4 NO 3  0.5 g/L-10 g/L, NaCl 0.5 g/L-20 g/L, polyethylene glycol 0.2 g/L-5 g/L, folic acid 0.08 g/L-2.56 g/L, VB1 0.01 g/L-0.8 g/L, indolebutyric acid 0.1 mg/L-4 mg/L, citric acid 0.01 g/L-0.8 g/L, pyruvic acid 0.05 g/L-4.5 g/L, arginine 0.1 g/L-7 g/L, lysine 0.1 g/L-8 g/L, leucine 0.02 g/L-0.5 g/L, aspartic acid 0.05 g/L-9 g/L, glutamic acid 1 μmol/L-100 μmol/L, betaine 50 mmol/L-250 mmol/L, histidine 0.1 mmol/L-3 mmol/L, cysteine 2 mmol/L-45 mmol/L, methionine 3 mmol/L-45 mmol/L, tween 0.5 g/L-50 g/L, span 0.2 g/L-10 g/L, chitosan 0.2 g/L-0.4 g/L, Fluconazole 2 mg/L-80 mg/L, Miconazole 0.5 mg/L-50 mg/L, Ketoconazole 0.5 mg/L-50 mg/L, ethylenediaminetetraacetic acid (EDTA) 0.05 g/L-0.5 g/L, isopropyl alcohol 0.5%-2% (V/V) and dimethyl sulfoxide 0.5%-2% (V/V) in specified amounts is added into the fermentation basal medium. 
     
     
       11. The method according to  claim 1 , wherein the temperature of fermentation is adjusted to 25-31° C. in the process of fermentation in step (b). 
     
     
       12. The method according to  claim 1 , wherein the pH of the fermentation broth is adjusted to 4.8-6.3 in the process of fermentation in step (b). 
     
     
       13. The method according to  claim 1 , wherein the pressure is adjusted to 0.05-0.1 Mpa and the dissolved oxygen is adjusted to 15-30% in the process of fermentation in step (b). 
     
     
       14. The method according to  claim 1 , comprising the steps of:
 (a) inoculating  Pleurotus ostreatus  strain CGMCC No.6232 into a seed medium, and culturing it at 25-28° C. for 3-5 days to prepare a seed liquor, wherein the seed medium comprises 25-40 g/L corn flour, 15-35 g/L soybean cake powder, 30-80 U/L α-amylase, 2-4.5 g/L KH 2 PO 4 , 0.2-3 g/L MgSO 4 .7H 2 O, and a balance of water; and 
 (b) inoculating the seed liquor into a fermentation basal medium with an inoculation amount of 4-20% (V/V), and culturing it at 25-31° C. for at least 6 days to obtain a fermentation broth of  Pleurotus ostreatus  mycelia, wherein the fermentation basal medium comprises 65-95 g/L glycerol, 40-80 g/L casein peptone, 2-4 g/L KH 2 PO 4 , 0.5-2 g/L MgSO 4 .7H 2 O, 7.5-15 mmol/L methionine, 7.5-15 mmol/L cysteine, and a balance of water, wherein ergothioneine is accumulated in mycelial cells of  Pleurotus ostreatus  mycelia; and 
 (c) extracting ergothioneine from the mycelial cells; 
 
       wherein using soybean cake powder as the nitrogen source in step (a) results in an increased amount of the production of ergothioneine in mycelial cells of said  Pleurotus ostreatus  strain CGMCC No.6232 in step (b) as compared to using soybean meal powder with the same concentration as nitrogen source of the seed medium. 
     
     
       15. The method according to  claim 1 , comprising the steps of:
 (a) inoculating  Pleurotus ostreatus  strain CGMCC No.6232 into a seed medium, and culturing it at 25° C. for 4 days to prepare a seed liquor, wherein the seed medium comprises 30 g/L corn flour, 15 g/L soybean cake powder, 80 U/L α-amylase, 3 g/L KH 2 PO 4 , 1.5 g/L MgSO 4 .7H 2 O, and a balance of water; and 
 (b) inoculating the seed liquor into a fermentation basal medium with an inoculation amount of 5% (V/V) and culturing it at 25° C. for 3 days, and then adjusting the pH to 6.0 and culturing it at 31° C. for 12 days to obtain a fermentation broth of  Pleurotus ostreatus  mycelia, wherein the fermentation basal medium comprises 75 g/L glycerol, 50 g/L casein peptone, 3 g/L KH 2 PO 4 , 1.5 g/L MgSO 4 .7H 2 O, 14 mmol/L methionine, 7.5 mmol/L cysteine, and a balance of water, wherein ergothioneine is accumulated in mycelial cells of  Pleurotus ostreatus  mycelia; and 
 (c) extracting ergothioneine from the mycelial cells; 
 
       wherein using soybean cake powder as the nitrogen source in step (a) results in an increased amount of the production of ergothioneine in mycelial cells of said  Pleurotus ostreatus  strain CGMCC No.6232 in step (b) as compared to using soybean meal powder with the same concentration as nitrogen source of the seed medium.